The rational development of new antineoplastic agents directed against tubulin, a protein critical for cell division, requires greater understanding of the interactions between the polypeptide subunits of tubulin and its two tightly bound guanine nucleotides. Interactions of ribose-modified GDP and GTP analogs with tubulin were examined in a microtubule-associated protein-dependent polymerization system. Inhibitory effects correlated well with nucleotide affinity for the exchangeable site, but the ability of analogs to support polymerization had no relationship to their affinity for tubulin. Microtubules formed with nucleotide hydrolysis were more stable than those formed without hydrolysis. The analog guanosine 5 foot-0-(3-thiotriphosphate) was a potent inhibitor of tubulin polymerization and GTP hydrolysis. Improved methods for the preparation of microtubule-associated proteins were developed. A new plant-derived antimitotic drug with antitubulin activity, combretastatin, was described. Efforts to separate the Alpha and Beta subunits of tubulin continued.